Recognition of a pathogen's genetic material as "foreign" alerts the cell to the presence of an invader. At the same time, an unchecked immune reaction to host nucleic acid incorrectly targets healthy cells for destruction, leading to autoimmune disease. It is therefore critical to understand how our immune systems detect foreign DNA, as well as the control mechanisms that restrain them in the absence of a true threat. In this application, we demonstrate that the barrier to autointegration factor (BAF) is a novel DMA-specific host defense protein. We have identified BAF as an inhibitor of poxviral DNA replication and hypothesize that BAF responds to any cytoplasmic DNA. As outlined in our three Specific Aims, our primary goal is to examine how BAF contributes to the cellular recognition and response to foreign DNA. In the first aim, we will clarify our understanding of the mechanism(s) through which BAF can inhibit various stages of the poxviral lifecycle. Mutant forms of BAF, established cell lines that overexpress or have been depleted of BAF, and both wild-type vaccinia and temperature-sensitive viruses will be utilized. The second aim builds upon our recent observation that BAF relocalizes to discrete foci upon transfection of plasmid DNA, and retards expression of genes encoded by these plasmids, indicating that BAF targets multiple sources of DNA. We will employ a battery of approaches including immunofluorescence analysis, BAF:plasmid co- precipitation studies, and reporter gene assays to characterize the recruitment of BAF to foreign DNA and its inhibitory impact upon binding. The third aim will focus on two general themes: [1] understanding how BAF's ability to function as a DNA sensor is regulated by dynamic phosphorylation, and [2] determining whether BAF's repression of foreign DNA is accompanied by activation of, or intersection with, cellular signaling pathways. In sum, the studies outlined here will further our understanding of BAF as a novel host defense protein. Understanding and exploiting the host/pathogen interactions that accompany infection with variola, the etiological agent of smallpox and a potential bioterrorist threat, and monkeypox, an emerging pathogen, is of significant biomedical importance. Exploring BAF's response against foreign DNA will also illuminate events that occur during other types of infections or the development of autoimmune disease, and will be of relevance to the establishment of effective strategies for gene therapy and DNA vaccination.